2378
Mass Spectrometric Quantitation of Nicotine, Cotinine,
and 4-(Methylnitrosamino)-1-(3-Pyridyl)1-Butanol in Human Toenails
Irina Stepanov,1 Rachel Feuer,2 Joni Jensen,2 Dorothy Hatsukami,2 and Stephen S. Hecht1
1
The Cancer Center, University of Minnesota; 2Transdisciplinary Tobacco Use Research Center, Minneapolis, Minnesota
Abstract
Numerous studies have quantified total cotinine (the sum
of cotinine and cotinine-N-glucuronide) and total 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol [NNAL; the sum of
NNAL and its O- and N-glucuronides (NNAL-Glucs)] in
the urine and blood of smokers, smokeless tobacco users,
and nonsmokers exposed to environmental tobacco smoke.
Analysis of hair and nails has several advantages over
blood and urine testing, such as accumulation of xenobiotics during long-term exposure, ease of collection, and
indefinite stability of samples. We developed sensitive
methods for quantitation of nicotine, cotinine, and NNAL
in human toenails. Nicotine and cotinine were analyzed by
gas chromatography-mass spectrometry-selected ion monitoring. NNAL was assayed using liquid chromatographyelectrospray ionization-tandem mass spectrometry-selected
reaction monitoring. The detection limits of the methods
were 0.01 ng/mg toenail for nicotine, 0.012 ng/mg toenail for
cotinine, and 0.02 pg/mg toenail for NNAL. In 35 smokers,
the mean nicotine level was 5.9 F 5.6 ng/mg toenail, mean
cotinine was 1.6 F 1.3 ng/mg toenail, and mean NNAL was
0.41 F 0.67 pg/mg toenail. Samples collected from six
nonsmokers were negative for NNAL. In smokers, NNAL
correlated with cotinine (r = 0.77; P < 0.0001). The results of
this study for the first time show the presence of cotinine
and NNAL in human toenails. These sensitive and
quantitative methods should be useful in epidemiologic
studies of the role of chronic tobacco smoke exposure,
including environmental tobacco smoke exposure, in human cancer. (Cancer Epidemiol Biomarkers Prev 2006;
15(12):2378 – 83)
Introduction
Cigarette smoking is a major cause of cancer deaths worldwide
and also causes vascular and respiratory diseases (1). Nicotine
and the tobacco-specific nitrosamine 4-(methylnitrosamino)-1(3-pyridyl)-1-butanone (NNK) are two important tobacco
smoke constituents representing its critical biological properties. Nicotine is the major tobacco alkaloid and the main
known addictive component of tobacco smoke. NNK is a
highly effective lung carcinogen in rats and also causes lung
tumors in mice and hamsters (2). It also produces tumors of the
pancreas, liver, and nasal mucosa (2), and administration
together with another tobacco-specific nitrosamine N¶-nitrosonornicotine caused tumors of the oral cavity in rats (3). NNK
and N¶-nitrosonornicotine have been classified recently by the
IARC as carcinogenic to humans (group 1; ref. 4).
Biomarkers of tobacco smoke exposure are crucial in
understanding mechanisms by which tobacco products cause
cancer. Nicotine is rapidly metabolized in the human body,
about 70% to 80% of the dose being converted to cotinine (5).
Measurements of nicotine and cotinine levels in urine, saliva,
and blood are commonly used to monitor systemic exposure to
cigarette smoke in smokers and nonsmokers exposed to
environmental tobacco smoke (ETS; refs. 6-9). Urinary metabolites of NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol
(NNAL) and its glucuronides (NNAL-Glucs), are excellent
biomarkers of NNK uptake in rodents and humans (10, 11).
Numerous studies have quantified total NNAL, the sum of
NNAL and NNAL-Glucs, in the urine of smokers, smokeless
Received 4/6/06; revised 9/25/06; accepted 10/10/06.
Grant support: National Cancer Institute.
The costs of publication of this article were defrayed in part by the payment of page charges.
This article must therefore be hereby marked advertisement in accordance with 18 U.S.C.
Section 1734 solely to indicate this fact.
Requests for reprints: Irina Stepanov, The Cancer Center, University of Minnesota, Mayo Mail
Code 806, 420 Delaware Street Southeast, Minneapolis, MN 55455. Phone: 612-624-4998;
Fax: 612-626-5135. E-mail: [email protected]
Copyright D 2006 American Association for Cancer Research.
doi:10.1158/1055-9965.EPI-06-0265
tobacco users, and nonsmokers exposed to ETS (11-16). Total
NNAL has also been quantified in the blood of smokers and
smokeless tobacco users (15, 16), and a practical and sensitive
method for total NNAL quantitation in plasma has been
developed recently (17).
Analysis of keratinic matrices, such as hair and nails, has
several advantages over urine, blood, and saliva testing. Thus,
growth rates are f1 cm monthly for hair (18), f0.3 cm
monthly for fingernails, and f0.1 cm monthly for toenails
(19); therefore, biomarker levels in hair and nails reflect
exposure over a longer period. Other advantages include
relative ease of sample collection and storage and seemingly
indefinite stability of the collected sample caused by incorporation of the analytes of interest into the keratinic matrix (19).
In recent years, hair was most commonly used for analysis of
drugs of abuse for forensic purposes (19-21). Hair analysis has
also been widely used to assess fetal exposure to nicotine
through maternal smoking during pregnancy (21-23) and
childhood exposure to ETS (18, 24, 25). Fingernails and
toenails have been used for the detection of drugs of abuse
(26) and in studies of arsenic intoxication (27, 28), occupational
exposures (29, 30), and environmental exposure of children to
trace elements (31-33). Unlike hair, nails grow in two
directions, length and thickness, thus leading to dual
mechanisms of xenobiotic incorporation, via the nail matrix
and nail bed (19, 34). Due to the contribution of the nail bed to
the incorporation process, the content of analytes in nail
clippings reflects cumulative exposure over a relatively long
period (19). Thus, nails seem to be more suitable in studies of
chronic exposure, whereas hair is useful in monitoring of
exposure during discrete periods by the axial distribution of
analytes. Toenails grow more slowly than fingernails and are
less likely to be environmentally contaminated with the analyte
of interest than fingernails or hair (19).
It is biologically plausible that cotinine and NNAL are
present in human toenails and could serve as biomarkers of
chronic human exposure to nicotine and NNK. Verghese et al.
Cancer Epidemiol Biomarkers Prev 2006;15(12). December 2006
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Cancer Epidemiology, Biomarkers & Prevention 2379
Materials and Methods
Caution. NNAL is carcinogenic and mutagenic and should
be handled with extreme care, using appropriate protective
clothing and ventilation at all times.
Figure 1. Structures of compounds analyzed in human toenails.
(35) reported an interesting physical sign related to smoking
cessation, which they termed ‘‘harlequin nail.’’ They observed
a distinct demarcation line in toenails of patients showing
when they had stopped smoking. In a recent study,
Al-Delaimy et al. (36) quantified nicotine in toenails using
high-performance liquid chromatography (HPLC) with electrochemical detection. It was found that toenail nicotine levels
differ significantly according to tobacco smoke exposure, being
f6-fold higher in smokers compared with nonsmokers
exposed to ETS. In his study, Al-Delaimy et al. used toenail
clippings collected as a part of the Nurses’ Health Study,
which was established in 1976 and involved 121,700 U.S.
female registered nurses. The same study provided toenail
clippings for investigation of toenail selenium concentrations
and bladder cancer risk (37). Toenail clippings are often
collected and stored in large-scale epidemiologic studies and
could be used for investigations of tobacco smoke constituent
uptake and development of cancer later in life.
The purpose of our study was to develop sensitive methods
for quantitation of cotinine and NNAL in human toenails.
Because HPLC with electrochemical detection is not as specific
as mass spectrometry (MS), we developed a gas chromatography-MS (GC-MS) method for quantitation of nicotine and
cotinine in toenails. NNAL was quantified by liquid chromatography-electrospray ionization-tandem MS (LC-ESI-MS/
MS). Structures of the biomarkers analyzed here are shown
in Fig. 1.
Chemicals and Enzymes. NNAL was purchased from
Toronto Research Chemicals, Inc. (Toronto, Ontario, Canada).
[Pyridine-D4]NNAL was synthesized from [pyridine-D4]ethyl
nicotinate (Cambridge Isotope Laborotories, Cambridge, MA)
as described previously (38, 39). [13C6]NNAL was synthesized
by NaBH4 reduction of [13C6]NNK (Cambridge Isotope
Laborotories). Nicotine, [CD3]nicotine, cotinine, [CD3]cotinine,
and h-glucuronidase (type IX-A from Escherichia coli) were
purchased from Sigma Chemical Co. (St. Louis, MO).
Apparatus. Analysis of nicotine and cotinine by GC-MSselected ion monitoring was carried out with a model 6890 GC
equipped with an autosampler and interfaced with a model
5973 mass-selective detector (Agilent Technologies, Palo Alto,
CA) as described previously (40, 41).
LC-ESI-MS/MS was carried out on a Finnigan TSQ
Quantum Discovery Max instrument (Thermo Electron Corp.,
Waltham, MA) interfaced with an Agilent model 1100 capillary
HPLC system and a model 1100 micro autosampler (Agilent
Technologies) by using a slight modification of a method
described previously (17). The HPLC was fitted with a 150 0.5 mm ZORBAX SB C18 RR 3.5-Am column (Agilent
Technologies) eluted in isocratic mode with 35% methanol in
H2O for 15 min at a flow rate of 10 AL/min. The column was
maintained at 25jC. MS/MS variables were as follows:
positive ion electrospray mode with selected reaction monitoring for m/z 210 ! 180 for NNAL, m/z 214 ! 184 for
[pyridine-D4]NNAL, and m/z 216 ! 186 for [13C6]NNAL, at
1.0 a.m.u. scan width. The collision gas was Ar at a pressure of
1 mTorr, with collision energy of 12 eV. The quadrupoles were
operated at a resolution of 0.7 a.m.u.
Figure 2. Analytic procedure for
nicotine, cotinine, and NNAL analysis in human toenails.
Cancer Epidemiol Biomarkers Prev 2006;15(12). December 2006
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2380 Nicotine, Cotinine, and NNAL in Toenails
Table 1. Nicotine and cotinine in subjects with different
exposures
Group
Nonsmokers,
not exposed to ETS
Nonsmokers,
exposed to ETS
Smokers
No. subjects*
Mean nicotine
(ng/mg)
Mean cotinine
(ng/mg)
4
0.11
0.012
2
0.37
0.036
2
1.2
0.33
*Samples from each subject were analyzed in duplicate.
Subjects. Thirty-five active smokers were recruited from a
longitudinal study of tobacco biomarkers and several smoking
cessation studies conducted at the Transdisciplinary Tobacco
Use Research Center (Minneapolis, MN). The entrance criteria
of these studies required subjects to smoke at least 10 cigarettes
daily for at least 1 year. Subjects were offered the opportunity
to participate in this addendum study for additional payment.
All studies were approved by the University of Minnesota
Research Subjects’ Protection Programs Institutional Review
Board Human Subjects Committee.
to pH 7). The mixture was applied to a 5-mL ChemElut
cartridge (Varian, Harbor City, CA) and eluted with 2 8 mL
CH2Cl2 into a 15-mL glass centrifuge tube. The combined
eluants were concentrated to dryness (Speedvac concentrator).
The dry residue was redissolved in 1 mL H2O, adjusted to pH
2 to 3, further purified on Oasis MCX cartridges (Waters Corp.,
Milford, MA), and transferred to autosampler vials as
described previously (12). Dry samples were stored at 20jC.
Before analysis by LC-ESI-MS/MS, samples were redissolved
in 10 AL of 2% methanol in H2O containing 2.5 pg/AL
[13C6]NNAL as injection standard, and 5 AL were injected.
Statistical Analyses. Pearson product moment correlation
coefficients and statistical significance of the correlations were
determined using SigmaPlot 2001, version 7.101 (SPSS, Inc.,
Chicago, IL).
Results
The procedures for nicotine, cotinine, and NNAL analysis in
toenails are summarized in the scheme shown in Fig. 2.
Nicotine and Cotinine Analysis. To analyze nicotine and
cotinine, toenails were digested at 50jC overnight in 0.5 mL
1 N NaOH. The next day, 5 AL of a mixture containing 5 ng/AL
each of [CD3]nicotine and [CD3]cotinine as internal standards
were added to a 5-mL glass centrifuge tube (Kimble, Vineland,
NJ) containing 0.5 mL of 25% aqueous K2CO3 and 1 mL
CH2Cl2. The digested toenail sample was added to the tube,
and the mixture was treated and analyzed as described
previously (40, 41).
Nicotine and Cotinine Assay. We initially analyzed only a
few samples collected from nonsmokers not exposed to ETS,
two nonsmokers exposed to ETS, and two smokers. The results
of this first experiment summarized in Table 1 were in good
agreement with the results reported by Al-Delaimy et al. (36)
in terms of nicotine levels in different exposure categories.
A typical GC-MS chromatogram of cotinine in human toenails
is shown in Fig. 3.
Precision of the developed nicotine and cotinine assay was
determined by dividing a toenail digest from a single smoker
into six aliquots and analyzing each for nicotine and cotinine.
Relative SDs were 10.1% for nicotine and 4.8% for cotinine.
The accuracy of the assay was determined by spiking an
ETS-exposed nonsmoker’s toenail digest with 0.5, 1, 2.5, and 5
ng cotinine/mg toenail. Analysis gave 0.42, 0.84, 2.3, and 4.4 ng
cotinine/mg toenail, respectively, producing good correlation
between spiked and measured cotinine (r = 0.99). The
detection limits of the method were 0.01 ng/mg toenail for
nicotine and 0.012 ng/mg toenail for cotinine.
NNAL Analysis. To analyze NNAL, toenails were digested at
50jC overnight in 1 mL 1 N NaOH. The next day, the digests
were added to a 15-mL glass centrifuge tube containing 10 pg
[pyridine-D4]NNAL as internal standard in 4 mL potassium
phosphate buffer (prepared from 0.1 mol/L KH2PO4 adjusted
NNAL Assay. The aqueous toenail digest was enriched
by partitioning with CH2Cl2 on a ChemElut liquid-liquid
extraction cartridge (Fig. 2). Final enrichment was accomplished by a mixed mode cation exchange extraction on an
Oasis MCX solid-phase extraction cartridge. The fraction
Analyses. Toenail clippings (20-30 mg for nicotine/cotinine
assay and 50-80 mg for NNAL assay) were weighed into 5-mL
polypropylene tubes and washed without agitation for 1.5 to
2 h in 2 mL CH2Cl2 at room temperature. Then, the tubes were
vortexed, CH2Cl2 was removed by aspiration, and the toenails
were vortexed one more time with 1 mL CH2Cl2. After
washing, the toenails were dried in a heating block at 50jC
(5-10 min).
Figure 3. GC-MS chromatograms
obtained on analysis of cotinine in
toenails from a smoker. A. Cotinine, m/z 176 (molecular ion) and
m/z 98 (used for quantitation). B.
[CD3]cotinine (internal standard),
m/z 179 (molecular ion), and m/z
101 (used for quantitation).
Cancer Epidemiol Biomarkers Prev 2006;15(12). December 2006
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Cancer Epidemiology, Biomarkers & Prevention 2381
Figure 4. Chromatograms obtained
on LC-ESI-MS/MS analysis of
NNAL, [pyridine-D4]NNAL (internal standard), and [13C6]NNAL
(injection standard). A. standard
mix. B. a smoker’s toenail sample.
containing NNAL was then directly analyzed by LC-ESI-MS/
MS. A typical chromatogram is shown on Fig. 4.
The detection limit of the assay for NNAL in toenails from
smokers was 0.02 pg/mg toenail, starting with a 50 mg sample.
Precision of the assay was determined by analyzing six
aliquots of a smoker’s digested toenail sample. The results
were 1.4 F 0.16 fmol NNAL/mg toenail (relative SD, 11.4%).
NNAL in the toenails from the same smoker was also
determined by a modified method, in which h-glucuronidase
treatment (37jC, overnight) was added after digestion and pH
adjustment to 6 to 7. This modification did not affect the
measured NNAL. The accuracy of the assay was determined
by spiking aliquots of pooled nonsmokers’ toenail digest with
0.5, 1, 2, 5, and 10 pg NNAL. Analysis gave 0.51, 1.1, 2.0, 5.3,
and 9.2 pg NNAL, respectively, producing high correlation
(r = 0.99) between spiked and measured NNAL. Recoveries of
internal standard averaged 22 F 10% (n = 28).
Analysis of Toenail Samples from Smokers. The assays
were applied to toenail samples from 35 smokers and 6
nonsmokers. Data for nicotine, cotinine, and NNAL in toenails
from smokers are summarized in Fig. 5. All nonsmoker
samples were negative for NNAL; nicotine and cotinine levels
in these subjects averaged 0.09 and 0.01 ng/mg, respectively.
Figure 5. Nicotine, cotinine, and NNAL in toenails of smokers.
Boxes, 25th and 75th percentiles; lines outside boxes, 10th and 90th
percentiles; dots, outliers.
In smokers, mean nicotine level was 5.9 F 5.6 ng/mg toenail,
mean cotinine was 1.6 F 1.3 ng/mg toenail, and mean NNAL
was 0.41 F 0.67 pg/mg toenail. NNAL correlated with cotinine
(r = 0.77; P < 0.0001; Fig. 6).
Discussion
We developed sensitive methods for quantitation of nicotine,
cotinine, and NNAL in human toenails. Previous studies on
cotinine and total NNAL in biological fluids, such as urine and
blood, provided information on short-term exposure to
nicotine and NNK. Thus, cotinine has a half-life of about 15
to 17 h, and the distribution half-life for urinary NNAL is 3 to 4
days. Toenail cotinine and NNAL levels integrate tobacco
smoke exposure over a relatively long period, about 3 to 5
months, thus being potentially useful biomarkers in studies of
the role of chronic tobacco smoke exposure in human cancer.
For the purposes of this study, the measurements were carried
out mainly in smokers; cotinine was also quantified in two
nonsmokers exposed to ETS. The limit of detection of these
assays reached into the femtomol range for nicotine and
cotinine and subfemtomol range for NNAL.
Figure 6. Relationship between cotinine and NNAL in the toenails of
31 smokers (r = 0.77; P < 0.0001).
Cancer Epidemiol Biomarkers Prev 2006;15(12). December 2006
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2382 Nicotine, Cotinine, and NNAL in Toenails
For nicotine and cotinine analysis in toenails, we used GCMS-selected ion monitoring, which is characterized by high
sensitivity and specificity. The method also allows using
deuterium-labeled nicotine and cotinine as internal standards,
providing high accuracy of nicotine and cotinine quantitation
in toenails. Overall, our results are in good agreement with
those reported by Al-Delaimy et al. (Table 1; ref. 36). Small
sample sizes limit our ability to compare these two methods,
and future studies are necessary to assess toenail nicotine and
cotinine levels in different exposure categories.
The method for NNAL analysis in toenails (Fig. 4) consists
of a few simple steps and resembles our recently published
method for total NNAL analysis in blood (17). Isocratic elution
of the LC column considerably reduced the time needed for
LC-MS-ESI analysis of a single sample. This modification did
not result in appearance of coeluting peaks, which would
interfere with quantitation (Fig. 5). It is unknown whether
NNAL-Glucs are present in toenails. NNAL-N-Gluc would be
hydrolyzed to NNAL at the initial step of the procedure, when
the toenails are digested in 1 N NaOH (42). NNAL-O-Gluc is
not hydrolyzed to NNAL by base treatment (42), but it is
converted to NNAL by treatment with h-glucuronidase
(12, 17, 42). Overnight treatment with h-glucuronidase of a
smoker’s toenail digest adjusted to pH 6 to 7 did not alter the
measured NNAL, thus indicating that there is no NNAL-OGluc present in toenails, or its amount is extremely low.
Although toenail nicotine levels were assessed previously
(36) and nicotine and cotinine were analyzed in another
keratinic matrix, hair (reviewed in ref. 43), no other study has
reported the presence of cotinine and NNAL in toenails. To
validate our new methods and investigate the relationship
between the analytes, we analyzed toenail clippings collected
from active smokers. The interindividual variability and the
ranges of toenail nicotine and cotinine levels observed here are
remarkably similar to those observed on hair analysis. Thus,
Kintz (44) reported 0.91 to 38.27 ng nicotine/mg hair and 0.09
to 4.99 ng cotinine/mg hair in 56 smokers. In our study,
nicotine ranged from 0.79 to 22.67 ng/mg toenail, and cotinine
ranged from 0.23 to 6.38 ng/mg toenail. This similarity is not
surprising because the growth of both hair and nail is slow, and
nicotine and cotinine are brought into these keratinic matrices
by similar mechanisms (through the blood circulation). However, in our opinion, toenail analysis has several advantages
over hair testing for studies of chronic exposure. Toenails are
considered to be relatively free of external contamination and
grow more slowly than hair, and the content of biomarkers in
nail clippings reflects cumulative rather than discrete exposure,
thus eliminating the issue of irregular growth and nonuniform
axial distribution of biomarkers present in hair. Moreover,
the limit of detection of nicotine in toenails (0.01 ng/mg toenail)
is lower than that reported for mass spectrometric analysis
of nicotine in hair (0.24 ng/mg hair; ref. 45).
The ratio between cotinine and NNAL in smokers’ toenails
was found to be f4,600 and is similar to that observed in the
urine of smokers (15). The correlation of toenail cotinine with
toenail NNAL observed in this study was also similar to that
reported for the urine of smokers (r = 0.68; ref. 46) and ETSexposed nonsmokers (r = 0.71; ref. 14).
Although this study provides accurate and sensitive
methods for nicotine, cotinine, and NNAL analysis in human
toenails, additional studies, such as a longitudinal study in
smokers and investigation of the kinetics of cotinine and
NNAL disappearance from quitters’ toenails, are necessary to
validate toenail cotinine and NNAL as useful biomarkers of
human chronic exposure to tobacco smoke.
In summary, the results of this study show for the first time
that cotinine and NNAL are present in human toenails. The
methods developed here should be useful in epidemiologic
studies of the role of chronic tobacco smoke exposure,
including ETS exposure, in human cancer.
Acknowledgments
We thank Steven G. Carmella for valuable advice on development of
method, Dr. Peter W. Villalta for help with MS techniques, and Bob
Carlson for editorial assistance.
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
IARC. Tobacco smoke and involuntary smoking. IARC monographs on the
evaluation of carcinogenic risks to humans. Vol. 83. Lyon (France): IARC;
2004. p.59 – 94.
Hecht SS. Biochemistry, biology, and carcinogenicity of tobacco-specific
N -nitrosamines. Chem Res Toxicol 1998;11:559 – 603.
Hecht SS, Rivenson A, Braley J, et al. Induction of oral cavity tumors in
F344 rats by tobacco-specific nitrosamines and snuff. Cancer Res 1986;46:
4162 – 6.
IARC. Smokeless tobacco and tobacco-specific nitrosamines. IARC monographs on the evaluation of carcinogenic risks to humans. Vol. 89. Lyon
(France): IARC. In press 2006.
Benowitz NL, Jacob P III. Metabolism of nicotine to cotinine studied by a
dual stable isotope method. Clin Pharmacol Ther 1994;56:483 – 93.
Hukkanen J, Jacob P III, Benowitz NL. Metabolism and disposition kinetics
of nicotine. Pharmacol Rev 2005;57:79 – 115.
DeLeon J, Diaz FJ, Rogers T, et al. Total cotinine in plasma: a stable
biomarker for exposure to tobacco smoke. J Clin Psychopharmacol 2002;225:
496 – 501.
Jarvis MJ, Primatesta P, Erens B, et al. Measuring nicotine intake in
population surveys: comparability of saliva cotinine and plasma cotinine
estimates. Nicotine Tob Res 2003;5:349 – 55.
Heinrich J, Hoelscher B, Seiwert M, et al. Nicotine and cotinine in adults’
urine: the German environmental survey 1998. J Expo Anal Environ
Epidemiol 2005;151:74 – 80.
Hecht SS. Tobacco carcinogens, their biomarkers, and tobacco-induced
cancer. Nat Rev Cancer 2003;3:733 – 44.
Hecht SS. Human urinary carcinogen metabolites: biomarkers for investigating tobacco and cancer. Carcinogenesis 2002;23:907 – 22.
Carmella SG, Akerkar S, Richie JP, Jr., et al. Intraindividual and
interindividual differences in metabolites of the tobacco-specific lung
carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in smokers’ urine. Cancer Epidemiol Biomarkers Prev 1995;4:635 – 42.
Anderson KE, Carmella SG, Ming Ye, et al. Metabolites of a tobacco-specific
lung carcinogen in nonsmoking women exposed to environmental tobacco
smoke in their homes. J Natl Cancer Inst 2001;93:378 – 81.
Hecht SS, Ming Ye, Carmella SG, et al. Metabolites of a tobacco-specific lung
carcinogen in the urine of elementary school-aged children. Cancer
Epidemiol Biomarkers Prev 2001;10:1109 – 16.
Hecht SS, Carmella SG, Chen M, et al. Quantitation of urinary metabolites of
a tobacco-specific lung carcinogen after smoking cessation. Cancer Res 1999;
59:590 – 96.
Hecht SS, Carmella SG, Ye M, et al. Quantitation of metabolites of 4(methylnitrosamino)-1-(3-pyridyl)-1-butanone after cessation of smokeless
tobacco use. Cancer Res 2002;62:129 – 34.
Carmella SG, Han S, Villalta PW, et al. Analysis of total 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol in smokers’ blood. Cancer Epidemiol Biomarkers Prev 2005;1411:2669 – 72.
Mahoney GN, Al-Delaimy WK. Measurement of nicotine in hair by
reversed-phase high-performance liquid chromatography with electrochemical detection. J Chromatogr B Biomed Sci Appl 2001;753:179 – 87.
Palmeri A, Pichini S, Pacifici R, et al. Drugs in nails. Physiology,
pharmacokinetics, and forensic toxicology. Clin Pharmacokinet 2000;382:
95 – 110.
Pepin G, Gaillard Y. Concordance between self-reported drug use and
findings in hair about cocaine and heroin. Forensic Sci Int 1997;84:
37 – 41.
Klein J, Forman R, Eliopoluos C, et al. A method for simultaneous
measurement of cocaine and nicotine in neonatal hair. Ther Drug Monit
1994;16:67 – 70.
Jacqz-Aigrain E, Zhang D, Maillard G, et al. Maternal smoking during
pregnancy and nicotine and cotinine concentrations in maternal and
neonatal hair. Br J Obstet Gynaecol 2002;109:909 – 11.
Chan D, Caprara D, Blanchette P, et al. Recent developments in meconium
and hair testing methods for the confirmation of gestational exposures to
alcohol and tobacco smoke. Clin Biochem 2004;37:429 – 38.
Al-Delaimy WK, Crane J, Woodward A. Passive smoking in children: effect
of avoidance strategies at home as measured by hair nicotine levels. Arch
Environ Health 2001;562:117 – 22.
Woodruff S, Conway T, Edwards C, et al. Acceptability and validity of hair
collection from Latino children to assess exposure to environmental tobacco
smoke. Nicotine Tob Res 2003;53:375 – 85.
Pounds A, Pearson EF, Turner TD. Arsenic in fingernails. J Forensic Sci 1979;
19:165 – 73.
Engelhart DA, Jenkins AJ. Detection of cocaine analytes and opiates in nails
from postmortem cases. J Anal Toxicol 2002;26:489 – 92.
Kile ML, Houseman EA, Rodrigues E, et al. Toenail arsenic concentrations,
GSTT1 gene polymorphisms, and arsenic exposure from drinking water.
Cancer Epidemiol Biomarkers Prev 2005;1410:2419 – 26.
Cancer Epidemiol Biomarkers Prev 2006;15(12). December 2006
Downloaded from cebp.aacrjournals.org on June 17, 2017. © 2006 American Association for Cancer Research.
Cancer Epidemiology, Biomarkers & Prevention 2383
29. Gerhardsson L, Englyst V, Lundstrom NG, et al. Lead in tissues of deceased
lead smelter workers. J Trace Elem Med Biol 1995;9:136 – 43.
30. Morton J, Mason HJ, Ritchie KA, et al. Comparison of hair, nails, and urine
for biological monitoring of low level inorganic mercury exposure in dental
workers. Biomarkers 2004;91:47 – 55.
31. Wilhelm M, Hafner D, Lombeck I, et al. Monitoring of cadmium, copper,
lead, and zinc status in young children using toenails: comparison with
scalp hair. Sci Total Environ 1991;103:199 – 207.
32. Alexiou D, Koutselinis A, Manolidis C, et al. The content of trace elements (Cu, Zn, Fe, Mg) in fingernails of children. Dermatologica 1980;160:
380 – 2.
33. Kasperek K, Iyengar GV, Feinendegen LE, et al. Multielement analysis of
fingernail, scalp hair, and water samples from Egypt (a preliminary study).
Sci Total Environ 1982;22:149 – 68.
34. Norton LA. Incorporation of thymidine-methyl-H3 and glycine-2H3 in the
nail matrix and bed of humans. J Invest Dermatol 1971;56:61 – 8.
35. Verghese A, Krish G, Howe D, et al. The harlequin nail. A marker for
smoking cessation. Chest 1990;97:236 – 8.
36. Al-Delaimy WK, Mahoney GN, Speizer FE, et al. Toenail nicotine levels as a
biomarker of tobacco smoke exposure. Cancer Epidemiol Biomarkers Prev
2002;11:1400 – 4.
37. Michaud DS, De Vivo I, Morris JS, et al. Toenail selenium concentrations and
bladder cancer risk in women and men. Br J Cancer 2005;93:804 – 6.
38. Hecht SS, Lin D, Castonguay A. Effects of a-deuterium substitution on the
39.
40.
41.
42.
43.
44.
45.
46.
mutagenicity of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK).
Carcinogenesis 1983;4:305 – 10.
Hecht SS, Young R, Chen CB. Metabolism in the F344 rat of 4-(N-methyl-Nnitrosamino)-1-(3-pyridyl)-1-butanone, a tobacco specific carcinogen. Cancer
Res 1980;40:4144 – 50.
Hecht SS, Carmella SG, Chen M, et al. Quantitation of urinary metabolites of
a tobacco-specific lung carcinogen after smoking cessation. Cancer Res 1999;
59:590 – 6.
Stepanov I, Hecht SS. Tobacco-specific nitrosamines and their pyridine-N glucuronides in the urine of smokers and smokeless tobacco users. Cancer
Epidemiol Biomarkers Prev 2005;144:885 – 91.
Carmella SG, Le K, Upadhyaya P, Hecht SS. Analysis of N - and O glucuronides of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in
human urine. Chem Res Toxicol 2002;15:545 – 50.
Al-Delaimy WK. Hair as biomarker for exposure to tobacco smoke. Tob
Control 2002;11:176 – 82.
Kintz P. Gas chromatographic analysis of nicotine and cotinine in hair.
J Chromatogr 1992;580:347 – 53.
Marchei E, Durgbanshi A, Rossi S, et al. Determination of arecoline (areca
nut alkaloid) and nicotine in hair by high-performance liquid chromatography/electrospray quadrupole mass spectrometry. Rapid Commun Mass
Spectrom 2005;1922:3416 – 8.
Hecht SS. Tobacco smoke carcinogens and lung cancer. J Natl Cancer Inst
1999;91:1194 – 210.
Cancer Epidemiol Biomarkers Prev 2006;15(12). December 2006
Downloaded from cebp.aacrjournals.org on June 17, 2017. © 2006 American Association for Cancer Research.
Mass Spectrometric Quantitation of Nicotine, Cotinine, and
4-(Methylnitrosamino)-1-(3-Pyridyl)-1-Butanol in Human
Toenails
Irina Stepanov, Rachel Feuer, Joni Jensen, et al.
Cancer Epidemiol Biomarkers Prev 2006;15:2378-2383.
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